While chimeric antigen receptor (CAR) gene therapy was initially tried and abandoned for HIV about 15 years ago, it has recently shown increasing usage and promise for treating cancer. This project revisits this approach for HIV, addressing the key barriers that prevented its success against HIV in the past, including harnessing hematopoietic stem precursor cells (HSPCs) to generate functional and durable CAR T cells in vivo, and generating novel CARs based on broadly neutralizing antibodies (bNAbs) and CAR combinations to prevent viral escape. Our specific aims are: 1. To optimize engraftment of bNAb CAR-transduced HSPCs. CAR T cells would develop entirely in vivo starting from HSPCs, avoiding the caveats of ex vivo T cell expansion and transduction to provide a lifelong self-renewing source. Engraftment will be tested and optimized in a humanized mouse model that recapitulates T cell development (CD34-mice, which are immunodeficient mice reconstituted with blood stem cells from adult human donors). 2. To generate bi-specific combined bNAb CARs to reduce options for HIV-1 escape. Novel CARs will be designed using new bi-specific antibody technologies, creating bi-specific CARs. 3. To select CAR combinations with the best antiviral breadth in vivo. Dual combinations of bi-specific CARs will be challenged in vivo in the CD34- mouse model with artificially diverse HIV-1 quasispecies to determine combinations with effective containment, analogous to combination antiretroviral therapy. These studies will lay the groundwork for successful new approaches to treating HIV infection with CAR gene therapy, which will be a valuable component in future cure strategies.